On the identity of the high spin heme components of cytochrome c oxidase

Abstract

In oxidized, resting cytochrome c oxidase (EC 1.9.3.1) and under most conditions of partial reduction ⩽ 50% of the heme components are detected by EPR spectroscopy. When the enzyme is fully reduced in the presence of equimolar quantities of cytochrome c, anaerobic reoxidation by an excess of a chemical oxidant (ferricyanide, porphyrexide) produces intense high and low spin heme signals simultaneously. The time range in which maximal high spin signals are observed is 0.1–2 s after mixing. Under these conditions 35–50% of the total heme a is accounted for by the low spin heme signal and 35–40% by the high spin signals, with the rhombic component accounting for 30–35% of the total heme. It is concluded that under these conditions, the major portion of both heme components must be EPR detectable. Thus, if the generally accepted assignment of the low spin signal to cytochrome a is adopted, it follows that in the experiments described, cytochrome a 3 is represented in the rhombic high spin signal. The quantities of heme represented in the axial high spin signal are too small for a definitive assignment; these signals could originate from either heme. When after formation of high spin signals as described, O 2 is admitted, the rhombic signal is eliminated within 4 ms. In the presence of the strongest rhombic high spin signals, the absorption band at 655 nm is only ⩽ 25% developed. The implications of these findings are discussed in the context of present hypotheses concerning the state and interactions of cytochrome c oxidase components during oxidation-reduction.